Our objective is to determine experimentally the pathway of folding of small, single-domain proteins. The method is to identify and characterize the structures of kinetic intermediates in the reversible unfolding/refolding transitions of these proteins (disulfide bonds intact). Most of the work is being done with bovine pancreatic ribonuclease A (RNase A) and with its derivative RNase S. The results are being tested with other small proteins to see if the conclusions can be generalized. RNase A, after unfolding, contains a mixture of two forms: a fast-folding and a slow-folding form. Recently we have shown that the slow-folding form is produced from the fast-holding form by the cis-trans isomerization of the proline residues in the unfolded molecule, as proposed earlier by Brandts and co-workers. We have found that two structural folding intermediates are well populated in the refolding of the major slow-folding species. The first intermediate is an early folding intermediate which has been detected by a 3H-labeling procedure involving the exchange with solvent of 3H-labeled amide protons of the polypeptide backbone: only those protons which are protected by H bonds or other structures are protected from exchange until they are trapped by the final folding process. The results suggest that the labeling procedure detects those H bonds in the early folding intermediate which are either conserved or reshuffled rapidly during folding. The second intermediate is a quasi-native intermediate which appears folded by tyrosine absorbance but which contains one or more "wrong" proline residues and is less stable than native RNase A. Our current work is focused on characterizing the structures of these two intermediates.